Peroxidase-like activities of iron(III)—Porphyrins: kinetics of the reduction of a peroxidatically active derivative of deuteroferriheme by anilines

The kinetics of the reduction by aniline and a series of substituted anilines of a peroxidatically active intermediate, formed by oxidation of deuteroferriheme with hydrogen peroxide, have been studied by stopped-flow spectrophotometry. The reaction with aniline was first order with respect to [intermediate] and showed first-order saturation kinetics with respect to [aniline]. The second-order rate constant was 2.0 ± 0.2 × 10⁵ M^?1 sec^?1 at 25°C (independent of pH in the range 6.60–9.68) compared with the value of 2.4 × 10⁵ M^?1 sec^?1 for the reaction of aniline with horseradish peroxidase Compound I. The effect of aniline substituents upon reactivity towards the heme intermediate closely paralled those reported for reaction with the enzymic intermediate. Anilines bearing electron-donating substituents reacted more rapidly and those bearing electron-withdrawing substituents more slowly than the unsubstituted amine. The rate constants for the heme intermediate reactions (k_dfh)found to be related to those for the enzymic reactions (k_hrp) by the equation:log k_DFH= 0.65log k_HRP+ 1.96 with a correlation coefficient of 0. 98.     

Hydrolysis of p-nitrotrifluoroacetanilide catalyzed by water and imidazole

The hydrolyses of p-nitrotrifluoroacetanilide catalyzed by water and imidazole were examined at 70°C. The pH-rate constant profile of the hydrolysis in H₂O was examined in the pH range 0.0–11.4. The hydrolysis was independent of pH in the region from pH 1.0 to 4.5, presumably a water-catalyzed reaction. The rate constant and the D₂O solvent isotope effect for this reaction were 1.0 × 10^?4 sec^?1 and 3.7, respectively. Both natural imidazole and imidazolium cation catalyzed hydrolysis. The rate constant of the hydrolysis catalyzed by neutral imidazole was determined to be 5.4 × 10^?3M^?1 sec^?1 and the D₂O solvent isotope effect was 1.8.     

Self-exchange rates and electron transfer kinetics of horse heart ferricytochrome C with amino acid-pentacyanoferrate(II) complexes

The electron transfer reactions of horse heart cytochrome c with a series of amino acid-pentacyanoferrate(II) complexes have been studied by the stopped-flow technique, at 25°C, μ = 0.100, pH 7 (phosphate buffer). A second-order behavior was observed in the case of the Fe(CN)₅ (histidine)^3? complex, with k = 2.8 x 10⁵ M^?1 sec^?1. For the Fe(CN)₅ (alanine)^4? and Fe(CN)₅(L-glutamate)^5? complexes, only a minor deviation of the second-order behavior, close to the experimental error (k = 3.2 × 10⁵ and 1.6 x 10⁵ M^?1 sec^?1, respectively) was noted at high concentrations of the reactants (e.g., 6 × 10^?4 M). The results are in accord with recent work on the Fe(CN)₆^4?/cytochrome c system demonstrating weak association of the reactants. The calculated self-exchange rate constants including electrostatic interactions for the imidazole,L -histidine, 4-aminopyridine, glycinate, β-alaninate, andL-glutamate pentacyanoferrate(II) complexes were 3.3 × 105, 3.3 × 10⁵, 2.8 × 10⁶,4.1 × 10²,5.5 × 10², and 6.0 M^?1 sec^?1, respectively. Marcus theory calculations for the cytochrome c reactions were interpreted in terms of two nonequivalent binding sites for the complexes, with the metalloprotein self-exchange rate constants varying from 10⁴ M^?1 sec^?1 (histidine, imidazole, and 4-aminopyridine complexes) to 10⁶ M^?1 sec ^?1 (glycinate, β-alaninate, and L-glutamate complexes).     

Mechanistic flexibility in the reduction of copper(II) complexes of aliphatic polyamines by mercapto amino acids

The reactions of copper(II)-ahphatic polyamine complexes with cysteine, cysteine methyl ester, penicillamine. and glutathione have been investigated, with the goal of understanding the relationship between RS^?-Cu(II) adduct structure and preferred redox decay pathway. Considerable mechanistic flexibility exists within this class of mercapto ammo acid oxidations, as changes in the rate law could be induced by modest variations in reductant concentration (at fixed [Cu(II)]_o), pH, and the structure of the redox partners. With excess cysteine present at 25°C, pH 5 0, I = 0 2 M (NaOAc), decay of 1:1 cys-S^?-Cu(II) transient adducts was found to be first order in both cys-SH and transient. Second-order rate constants characteristic of Cu(dien)²⁺ (6 1 × 10³M^?1sec^?1), Cu(Me₅dien)²⁺ (2.7 × 10³M^?1 sec^?1), Cu(en)₂²⁺ (2.1 × 10³M^?1 sec^?1), and Cu(dien)₂²⁺ (4.7 × 10³ M^?1 sec ^?1) are remarkably similar, considering substantial differences in the composition and geometry of the oxidant first coordination sphere. A mechanism involving attack of cysteine on the coordinated sulfur atom of the transient, giving a disulfide anion radical intermediate, is proposed to account for these results Moderate reactivity decreases in the cysteine-Cu(dien)²⁺, Cu(Me₅dien)²⁺ reactions with increasing [H⁺] (pH 4–6) reflect partial protonation of the polyamine ligands. A very different rate law, second order in the RS^?-Cu(II) transient and approximately zeroth order in mercaptan, applies in the pH 5.0 oxidations of cysteine methyl ester, penicillamine, and glutathione by Cu(dien)²⁺ and Cu(Me₅dien)²⁺. This behavior suggests the mtermediacy of di-μ-mercapto-bridged binuclear Cu(II) species, in which a concerted two-electron change yields the disulfide and Cu(I) products. Similar hydroxo-bridged intermediates are proposed to account for the transition from first- to second-order transient dependence in cysteine oxidations by Cu(dien)²⁺ and Cu(Me₅dien)²⁺ as the pH is increased from 5 to 7. Yet another rate law, second order in transient and first order in cysteine, applies in the pH 5.0 oxidation of cysteine by Cu(Me₆tren)²⁺ (k(25°C) 7.5 × 10⁷ M^?2 sec^?1, I = 0.2 M). Steric rigidity of this trigonal bipyramidal oxidant evidently protects the coordinated sulfur atom from attack in a RSSR^?-forming pathway. Formation of a coordinated disulfide in the rate-determining step is purposed, coupled with attack of a noncoordinated cysteine molecule on a vacated coordination position to stabilize the (Me₆(tren)Cu(I) product.     

Experimental determination of the redox potential of the superoxide radical O 2

The redox potential of ^?O₂^? was determined based on the dependence of the electron transfer reaction from ^?O₂^? upon the known redox potential of various acceptors A (including a range of quinones, dyes and ferricyanide). The efficiencies and the rates of these electron transfer processes were determined, using the technique of pulse radiolysis, by monitoring the formation kinetics of the semiquinone radical anions at the appropriate wavelength. From the percentage efficiency versus E^o′ plot, an E^o′ value of + 0.15 ± 0.01 V at pH 7.0 and 22°C, or E^o = + 0.57 ± 0.01 V, for the ${}^{\mathrm{?}}\text{O}{}_2{}^{\mathrm{?}}\text{O}{}_2$ couple was obtained. The rate k(^?O₂^? + A → O₂ + ^?A^?) = 9.8 × 10⁸ M^?1 sec^?1 where A = p-benzoquinone. The E^o value for the ^?HO₂ radical is > 1.0 V. It was also found that hydroquinone can quantitatively reduce ^?O₂^? to H₂O₂, k(^?O₂^? + QH₂ → QH^? + HO₂^?) = 1.6 ± 0.1 × 10⁷ M^?1 sec^?1 at pH 7.0 and 22°C.     

Radical scavenging and chemical repair of rutin observed by pulse radiolysis: as a basis for radiation protection

Abstract

Pulse radiolysis was conducted to investigate: several fundamental reactions of a natural flavonoid, rutin, and its glycosylated form (αG-rutin) as a basis for their radiation protection properties; the reactions with ^?OH (radical scavenging) and dGMP radical, dGMP^? (chemical repair), which was used as a model of initial and not yet stabilised damage on DNA. Three absorption peaks were commonly seen in the reactions of the flavonoids with ^?OH, showing that their reactive site is the common structure, i.e. aglycone. One among the three peaks was attributed to the flavonoid radical produced as a result of the removal of a hydrogen atom. The same peak was found in their reactions with dGMP^?, showing that dGMP^? is chemically repaired by obtaining a hydrogen atom supplied from the flavonoids. Such a spectral change due to the chemical repair was as clear as never reported. The rate constants of the chemical repair reaction were estimated as (9?±?2)×10⁸ M^?1 s^?1 and (6?±?1)×10⁸ M^?1 s^?1 for rutin and αG-rutin, respectively. The rate constants of the radical scavenging reactions towards ^?OH were estimated as (1.3?±?0.3)×10¹⁰ M^?1 s^?1 and (1.0?±?0.1)×10¹⁰ M^?1 s^?1 for rutin and αG-rutin, respectively. In addition, there was no obvious difference between rutin and αG-rutin, indicating that the glycosylation does not change early chemical reactions of rutin.     

Pulse radiolytically generated superoxide and Cu(II)-salicylates.

The rate constants of the reactions between pulse radiolytically produced superoxide anions and the Cu(II) chelates of salicylate, acetylsalicylate, p-aminosalicylate and diisopropylsalicylate were determined at pH 7.5 and found to range from 0.8 to 2.4 × 10⁹ M^?1 sec^?1. It was intriguing to note that they had a superoxide dismutase activity identical with that of native cuprein-copper (k₂₄₅ = 1.3 × 10⁹ M^?1 sec^?1 per g-atom of Cu). These measurements confirm our earlier observations using indirect assays that all copper salicylates act as perfect model superoxide dismutases and favour the proposal that the activity of anti-inflammatory agents might be assigned to their in vivo formed Cu complexes.     

A comparison of the binding of imidazole to valence hybrid and methemoglobin: an assignment of individual heme reactivity

The ferric hemes of valence hybrid hemoglobins combine with imidazole in a manner analogous with the hemes of methemoglobin. Equilibrium studies show that imidazole binding to methemoglobin is minimally described by the sum of two independent processes (K₁ = 200 M^?1 and K₂ = 37 M^?1), both of which contribute equally to the observed difference spectrum. Using valance hybrid hemoglobins, which show single binding processes under similar conditions, it is possible to identify the high affinity sites in methemoglobin with the α chains and the low affinity sites with the β chains.Kinetic studies show that the valance hybrid hemoglobins react in a single exponential fashion with imidazole in contrast with methemoglobin which shows a biphasic reaction (k₁ = 85 M^?1 sec^?1k₂ = 25 M^?1 sec^?1). A comparison of the rates of reaction of the hybrids allows the assignment of the fast phase in methemoglobin to the β chains and the slow phase to the α chains.The heterogeneity of the imidazole reaction with methemoglobin occurs over the pH range 5.5–9.5 within which two ionization processes are discernable at pH 6.9 and 7.5.     

Kinetics of Ni(II) and Co(II) monocomplex formation with O-phospho-dl-serine

The complexation reactions of O-phospho-DL-serine with Ni(II) or Co(II) were studied at 25°C and ionic strength 0.2 M (KNO₃) by temperature-jump. The observed rate constants for formation of the Ni²⁺ and Co⁺² monocomplexes were (1.32 ± 0.09) × 10⁵ and (1.73 ± 0.33) × 10⁷ M^?1 sec^?1, respectively. Complexation is postulated to involve formation of a monocoordinated steady state intermediate followed by rate-determining chelate ring closure.     

Cooperative nonenzymic base recognition. Kinetics of the binding of a base monomer to a complementary polynucleotide template

The kinetics of double-helix formation by poly U and the complementary monomer N-6,9-dimethyladenine (m⁶m⁹A) has been measured using a new fast temperature-jump apparatus. The cooperative binding kinetics are complicated by the extensive self-association of the monomers, but a satisfactory analysis using average relaxation times was possible in terms of three different models. Application of a model which considers only monomer binding yields the upper limit for the binding rate constant of an m⁶m⁹A monomer next to an already bound monomer on a poly U strand: (2 ± 0.4) × 10⁸ M^?1sec^?1. A lower limit is found by using a model which allows for binding of all m⁶m⁹A stacks to poly U with equal rate constants: (3 ± 0.3) × 10⁷ M^?1sec^?1. A third model with “weighted” rate constants consistent with the data: (7.5 ± 1.0) × 10⁷ M^?1sec^?1. The rate of cooperative binding of m⁶m⁹A to the trimer UpUpU has also been measured. The rate constants obtained with the trimer agree with those obtained with the polymer for each of the three models within experimental error.     

Kinetics of the stacking of ethidium bromide by the raman laser temperature-jump method

Raman laser temperature-jump measurements have been made on concentrated solutions of ethidium bromide. Two relaxations were observed. The faster has a lifetime of less than 30 ns and is attributed to rotation of the phenyl ring. The slower relaxation is concentration dependent and is due to the parallel stacking of two dye molecules. The forward and reverse rates for this process are (4.6 ± 1.4) × 10⁸ M^?1s^?1 and (6.7 ± 1.4)× 10⁶ s^?1, respectively, at 25°C. 0.25 M ionic strength, and pH 6.9. This reverse rate and those of three similar reactions are found to fit a linear free energy plot. The implications of these results for studies of nucleic acid base stacking are discussed.     

The reaction of cytochalasin a with sulfhydryl groups

The reaction of cytochalasin A with sulfhydryl groups was examined. Cysteine and glutathione reacted readily with cytochalasin A at pH 7.0, 20°C, following second-order kinetics with rate constants of 7,600 M^?1 sec^?1 and 870 × 10³ M^?1 sec^?1. No reaction of cytochalasin B could be demonstrated under the same conditions. The reaction of cytochalasin A with the amino group of glycine ethyl ester had a second-order rate constant of 0.02 M^?1 sec^?1. Cytochalasin A did not react with sufhydryl groups of native ovalbumin or lactic dehydrogenase but reacted with an least 2 and 12 groups respectively when the proteins were denatured in 0.1% SDS. The reactivity of cytochalasin A with sulfhydryl groups is attributable to the α,β-unsaturated ketone groups it contains.     

Kinetics and mechanism of catalase action. Formation of the intermediate complex

The kinetics of formation of the intermediate complex between catalase and H₂O₂ has been reexamined. It has been shown that the kinetics consists of a rapid and of a subsequent slow phase. At the maximum of the transient decrement of the optical absorption, the system was found to be in a terminal state with regard to the rapid phase. On this basis, the formation curve of the intermediate complex was calculated. From the parameters of the curve the maximal saturation of catalase hematins (from horse erythrocytes) by H₂O₂ is 35%. The absolute spectrum of the intermediate complex was established. The variation of the previously calculated rate constant of formation of the intermediate complex was shown to be due to the inapplicability of the pre-steady-state approximation to the rate data. By applying a more general approach and by the use of a computer, the individual rate constants of the peroxidatic scheme were calculated (relevant to micromolar solutions of catalase) k₁ = (3.0 ± 0.2) × 10⁶ M^?1 sec^?1k₄ = (5.6 ± 0.3) × 10⁶ M^?1 sec^?1 These values are 2.2 times higher in a nanomolar solution.     

Kinetics of oxidation of tyrosine by a model alkoxyl radical

Abstract

Oxidation of tyrosine moieties by radicals involved in lipid peroxidation is of current interest; while a rate constant has been reported for reaction of lipid peroxyl radicals with a tyrosine model, little is known about the reaction between tyrosine and alkoxyl radicals (also intermediates in the lipid peroxidation chain reaction). In this study, the reaction between a model alkoxyl radical, the tert-butoxyl radical and tyrosine was followed using steady-state and pulse radiolysis. Acetone, a product of the β-fragmentation of the tert-butoxyl radical, was measured; the yield was reduced by the presence of tyrosine in a concentration- and pH-dependent manner. From these data, a rate constant for the reaction between tert-butoxyl and tyrosine was estimated as 6?±?1 × 10⁷ M^?1 s^?1 at pH 10. Tyrosine phenoxyl radicals were also monitored directly by kinetic spectrophotometry following generation of tert-butoxyl radicals by pulse radiolysis of solutions containing tyrosine. From the yield of tyrosyl radicals (measured before they decayed) as a function of tyrosine concentration, a rate constant for the reaction between tert-butoxyl and tyrosine was estimated as 7?±?3 × 10⁷ M^?1 s^?1 at pH 10 (the reaction was not observable at pH 7). We conclude that reaction involves oxidation of tyrosine phenolate rather than undissociated phenol; since the pK_a of phenolic hydroxyl dissociation in tyrosine is ～ 10.3, this infers a much lower rate constant, about 3 × 10⁵ M^?1 s^?1, for the reaction between this alkoxyl radical and tyrosine at pH 7.4.     

Free radicals and singlet oxygen scavengers: Reaction of a peroxy-radical with β-carotene,diphenyl furan and 1,4-diazobicyclo(2,2,2)-octane

The ‘singlet oxygen scavengers’. 1,4-diazobicyclo(2,2,2)-octane (DABCO), diphenyl furan and β-carotene react rapidly with the organic peroxyradical CCl₃O₂^?. The absolute reaction rate constants k = 1.2 ± 0.2 × 10⁷, 6 ± 2 × 10⁷ at 1.5 ± 0.2 × 10⁹ M^?1s^?1 respectively have been determined by pulse radiolysis. Comparison with other data suggest that other free radicals are also likely to react with these compounds; in the case of the hydroxyl radical and DABCO k = 1.25 × 10⁹ M^?1s^?1 has been determined.     

Intramolecular participation of the amide group in the hydrolysis of p-nitrophenyl N-(bromoacetyl) anthranilate

The rate of hydrolysis of p-nitrophenyl N-(bromoacetyl) anthranilate (Ib) has been measured in aqueous solution between pH 1 and 6.5 has been found to increase linearly with pH at pH higher than 3. An abnormally large apparent alkaline rate constant of 3.8 × 10⁶M^?1 sec^?1 has been determined. Intramolecular nucleophilic displacement by the amide group at the carbonyl carbon of the ester occurred and a cyclic intermediate was formed. This intermediate has been detected by direct isolation and by measurements of the proton release accompanying the reaction. The rates of hydrolysis of analogous derivatives (IIb-IIIb-IV), for which this intramolecular assistance was not possible, were slower by a factor of about 5 × 10⁵. Such an example of intramolecular catalysis may be useful for a better understanding of the enzymatic catalysis.     

Trypsin inhibitors from ridged gourd (Luffa acutangula Linn.) seeds: Purification,properties, and amino acid sequences

Two trypsin inhibitors, LA-1 and LA-2, have been isolated from ridged gourd (Luffa acutangula Linn.) seeds and purified to homogeneity by gel filtration followed by ion-exchange chromatography. The isoelectric point is atpH 4.55 for LA-1 and atpH 5.85 for LA-2. The Stokes radius of each inhibitor is 11.4 å. The fluorescence emission spectrum of each inhibitor is similar to that of the free tyrosine. The biomolecular rate constant of acrylamide quenching is 1.0×10⁹ M^?1 sec^?1 for LA-1 and 0.8 × 10⁹ M^?1 sec^?1 for LA-2 and that of K₂HPO₄ quenching is 1.6×10¹¹ M^?1 sec^?1 for LA-1 and 1.2×10¹¹M^?1 sec^?1 for LA-2. Analysis of the circular dichroic spectra yields 40%α-helix and 60%Β-turn for La-1 and 45%α-helix and 55%Β-turn for LA-2. Inhibitors LA-1 and LA-2 consist of 28 and 29 amino acid residues, respectively. They lack threonine, alanine, valine, and tryptophan. Both inhibitors strongly inhibit trypsin by forming enzymeinhibitor complexes at a molar ratio of unity. A chemical modification study suggests the involvement of arginine of LA-1 and lysine of LA-2 in their reactive sites. The inhibitors are very similar in their amino acid sequences, and show sequence homology with other squash family inhibitors.     

Structural and physicochemical analysis of the reaction between the anti-lysozyme antibody D1.3 and the anti-idiotopic antibodies E225 and E5.2

The reaction between the mouse (BALB/c) anti-idiotiopic monoclonal antibodies E225 and E5.2 and idiotopes on the (BALB/c) anti-lysozyme monoclonal antibody D1.3 has been characterized by titration calorimetry, by equilibrium sedimentation and by the determination of binding association and dissociation rates. The reaction between E5.2 and D1.3 is driven by a large negative enthalpy and its rate and equilibrium association constants are comparable to those observed in other antigen–antibody reactions. In contrast, the reaction between E225 and D1.3 is entropically driven and characterized by slow association kinetic (1 × 10³ M^?1 sec^?1) and a resulting low equilibrium constant (K_a = 2 × 10⁵M ^?1). A correlation of these properties with the three-dimensional structure of the Fab225-FabD1.3 complex, previously determined by X-ray diffraction methods to 2.5 Å resolution, indicates that conformational changes of several D1.3 contacting residues, located in its complementarity determining regions, may explain these features of the reaction.     

Evaluation of the Antioxidant Actions of Ferulic Acid and Catechins

We have evaluated the abilities of ferulic acid, (±) catechin, (+) catechin and (-) epicatechin to scavenge the reactive oxygen species hydroxyl radical (OH±), hypochlorous acid (HOCl) and peroxyl radicals (RO₂).

Ferulic acid tested at concentrations up to 5 mM inhibited the peroxidation of phospholipid liposomes. Both (±) and (+) catechin and (-) epicatechin were much more effective. All the compounds tested reacted with trichloromethyl peroxyl radical (CCl₃O₂) with rate constants > 1 × 10⁶M^?1s^?1.

A mixture of FeCl₃-EDTA, hydrogen peroxide (H₂O₂) and ascorbic acid at pH 7.4, has often been used to generate hydroxyl radicals (OH.) which are detected by their ability to cause damage to the sugar deoxyribose. Ferulic acid, (+) and (±) catechin and (-) epicatechin inhibited deoxyribose damage by reacting with OH. with rate constants of 4.5 × 10⁹M^?1s^?1, 3.65 × 10⁹M^?1s^?1, 2.36 × 10⁹M^?1s^?1 and 2.84 × 10⁹M^?1s^?1 respectively. (-) Epicatechin, ferulic acid and the (+) and (±) catechins exerted pro-oxidant action, accelerating damage to DNA in the presence of a bleomycin-iron complex. On a molar basis, ferulic acid was less effective in causing damage to DNA compared with the catechins.

A mixture of hypoxanthine and xanthine oxidase generates O₂ which reduces cytochrome c to ferrocytochrome c. (+) Catechin and (-) epicatechin inhibited the reduction of cytochrome c in a concentration dependent manner. Ferulic acid and (±) catechin had only weak effects.

All the compounds tested were able to scavenge hypochlorous acid at a rate sufficient to protect alpha-1-antiproteinase against inactivation. Our results show that catechins and ferulic acid possess antioxidant properties. This may become important given the current search for “natural” replacements for synthetic antioxidant food additives.     

Equilibrium dialysis binding studies of 1,N6-ethenoadenosine diphosphate (epsilon ADP) to myosin, heavy meromyosin, and subfragment one

The binding of the fluorescent analog of adenosine diphosphate (ADP)¹, 1,N⁶-ethenoadenosine diphosphate (εADP) to myosin and its subfragments, heavy meromyosin (HMM) and subfragment one (S1), has been studied under analagous conditions to those previously used in comparable studies on the binding of ADP to these molecules. The results indicate that there are two binding sites for εADP on myosin and HMM, and one site on S1. The dissociation constants for all had an identical value, within experimental error, of 2.0 (^± .5) × 10^?5 M^?1. This is identical to the values found by Young (J. Biol. Chem., $\text{242}$, 2790 (1967)) for ADP. In addition, the kinetics of hydrolysis of εATP versus ATP by S1 were studied. Values of V_max and K_m were 25 μM phosphate sec^?1 (gm protein)^?1 and 5 × 10^?5 M^?1 for ATP, and 80 μN phosphate sec^?1 (gm protein)^?1 and 45 × 10^?5 M^?1 for εATP. The results indicate that the increased V_max that occurs when εATP is used as a substitute for ATP is not due to either an increased binding affinity of ATP for myosin and its subfragments, nor due to a decreased binding affinity of εATP versus ADP. This in turn suggests that the increase in V_max may be due to an increased hydrolytic rate of εATP vs ATP in the enzyme substrate complex.